scholarly journals Regulation of cyclin-dependent kinase (Cdk) 2 Thr-160 phosphorylation and activity by mitogen-activated protein kinase in late G1 phase

2000 ◽  
Vol 349 (3) ◽  
pp. 869-876 ◽  
Author(s):  
Mario CHIARIELLO ◽  
Eliana GOMEZ ◽  
J. Silvio GUTKIND
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Map Kinase ◽  
Map Kinases ◽  
Mitogen Activated Protein Kinase ◽  
G1 Phase ◽  
Cyclin Dependent Kinase ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Mitogen-activated protein (MAP) kinases, p42MAPK and p44MAPK, are central components of growth-promoting signalling pathways. However, how stimulation of MAP kinases culminates in cell-cycle progression is still poorly understood. Here we show that mitogenic stimulation of NIH 3T3 cells causes a sustained activation of MAP kinases, which lasts until cells begin progressing through the G1/S boundary. Furthermore, we observed that disruption of the MAP-kinase pathway with a selective MEK (MAP kinase/extracellular-signal-regulated protein kinase kinase) inhibitor, PD98059, prevents the activation of cyclin-dependent kinase (Cdk) 2 and DNA synthesis, even when added during late G1 phase, once the known mechanisms by which MAP kinase controls G1 progression, accumulation of G1 cyclins and degradation of Cdk inhibitors have already taken place. Moreover, we provide evidence indicating that MAP kinases control Cdk2 Thr-160 activating phosphorylation and function, possibly by regulating the activity of a Cdk-activating kinase, thus promoting the re-initiation of DNA synthesis. These findings suggest the existence of a novel mechanism whereby signal-transducing pathways converging on MAP kinases can affect the cell-cycle machinery and, ultimately, participate in cell-growth control.

scholarly journals Genetic Complementation Screen Identifies a Mitogen-activated Protein Kinase Phosphatase, MKP3, as a Regulator of Dopamine Transporter Trafficking

2008 ◽  
Vol 19 (7) ◽  
pp. 2818-2829 ◽  
Author(s):  
Ole Valente Mortensen ◽  
Mads Breum Larsen ◽  
Balakrishna M. Prasad ◽  
Susan G. Amara
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Dopamine Transporter ◽  
Map Kinases ◽  
Kinase Inhibitors ◽  
Mitogen Activated Protein Kinase ◽  
Genetic Complementation ◽  
Monoamine Transporters ◽  
Map Kinase Phosphatase ◽  
Mitogen Activated Protein

The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C–induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis.

scholarly journals A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

1997 ◽  
Vol 137 (2) ◽  
pp. 433-443 ◽  
Author(s):  
Xiao Min Wang ◽  
Ye Zhai ◽  
James E. Ferrell
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Map Kinases ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Mitogen Activated Protein Kinase ◽  
Mitotic Progression ◽  
Map Kinase Phosphatase ◽  
Mitogen Activated Protein ◽  
Map Kinase Kinase

The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.

scholarly journals 3pK, a novel mitogen-activated protein (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways.

1996 ◽  
Vol 16 (12) ◽  
pp. 6687-6697 ◽  
Author(s):  
S Ludwig ◽  
K Engel ◽  
A Hoffmeyer ◽  
G Sithanandam ◽  
B Neufeld ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Stress Responses ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Cascades ◽  
Mitogen Activated Protein ◽  
Stimulation Of

Recently we have identified a mitogen-activated protein kinase (MAPK)-activated protein kinase, named 3pK (G. Sithanandam, F. Latif, U. Smola, R. A. Bernal, F.-M. Duh, H. Li, I. Kuzmin, V. Wixler, L. Geil, S. Shresta, P. A. Lloyd, S. Bader, Y. Sekido, K. D. Tartof, V. I. Kashuba, E. R. Zabarovsky, M. Dean, G. Klein, B. Zbar, M. I. Lerman, J. D. Minna, U. R. Rapp, and A. Allikmets, Mol. Cell. Biol. 16:868-876, 1996). In vitro characterization of the kinase revealed that 3pK is activated by ERK. It was further shown that 3pK is phosphorylated in vivo after stimulation of cells with serum. However, the in vivo relevance of this observation in terms of involvement of the Raf/MEK/ERK cascade has not been established. Here we show that 3pK is activated in vivo by the growth inducers serum and tetradecanoyl phorbol acetate in promyelocytic HL60 cells and transiently transfected embryonic kidney 293 cells. Activation of 3pK was Raf dependent and was mediated by the Raf/MEK/ERK kinase cascade. 3pK was also shown to be activated after stress stimulation of cells. In vitro studies with recombinant proteins demonstrate that in addition to ERK, members of other subgroups of the MAPK family, namely, p38RK and Jun-N-terminal kinases/stress-activated protein kinases, were also able to phosphorylate and activate 3pK. Cotransfection experiments as well as the use of a specific inhibitor of p38RK showed that these in vitro upstream activators also function in vivo, identifying 3pK as the first kinase to be activated through all three MAPK cascades. Thus, 3pK is a novel convergence point of different MAPK pathways and could function as an integrative element of signaling in both mitogen and stress responses.

scholarly journals Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle

1999 ◽  
Vol 338 (3) ◽  
pp. 643-649 ◽  
Author(s):  
Soma RAKHIT ◽  
Ann-Marie CONWAY ◽  
Rothwelle TATE ◽  
Tara BOWER ◽  
Nigel J. PYNE ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Airway Smooth Muscle ◽  
Essential Role ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activation ◽  
Mitogen Activated Protein ◽  
Sphingosine 1 Phosphate ◽  
Stimulation Of

We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate that S1P activates c-Src and Grb-2–PI-3K (intermediates in the p42/p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is consistent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common permissive signal transduction pathway linked to DNA synthesis.

scholarly journals Insulin-Activated Protein Kinase Cβ Bypasses Ras and Stimulates Mitogen-Activated Protein Kinase Activity and Cell Proliferation in Muscle Cells

2000 ◽  
Vol 20 (17) ◽  
pp. 6323-6333 ◽  
Author(s):  
Pietro Formisano ◽  
Francesco Oriente ◽  
Francesca Fiory ◽  
Matilde Caruso ◽  
Claudia Miele ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Antisense Oligonucleotide ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Stimulation ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Stimulation Of

ABSTRACT In L6 muscle cells expressing wild-type human insulin receptors (L6hIR), insulin induced protein kinase Cα (PKCα) and β activities. The expression of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activation by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 (IRS-1) expression caused a 90% decrease in insulin-induced PKCα and -β activation and blocked insulin stimulation of mitogen-activated protein kinase (MAPK) and DNA synthesis. Blocking PKCβ with either antisense oligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by >80% but did not affect epidermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, blocking c-Ras with lovastatin or the use of the L61,S186 dominant negative Ras mutant inhibited insulin-stimulated MAPK activity and DNA synthesis by only about 30% but completely blocked the effect of EGF. PKCβ block did not affect Ras activity but almost completely inhibited insulin-induced Raf kinase activation and coprecipitation with PKCβ. Finally, blocking PKCα expression by antisense oligonucleotide constitutively increased MAPK activity and DNA synthesis, with little effect on their insulin sensitivity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKCα and -β. (ii) IRS-1 phosphorylation is necessary for insulin activation of these PKCs in the L6 cells. (iii) In these cells, PKCβ plays a unique Ras-independent role in mediating insulin but not EGF or other growth factor mitogenic signals.

scholarly journals Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice, but not in hepatocytes from p16INK4a-null mice

1998 ◽  
Vol 336 (3) ◽  
pp. 551-560 ◽  
Author(s):  
Kelly L. AUER ◽  
Jong-Sung PARK ◽  
Prem SETH ◽  
Robert J. COFFEY ◽  
Gretchen DARLINGTON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Primary Hepatocytes ◽  
Rhoa Gtpase ◽  
Mapk Signalling ◽  
Mitogen Activated Protein

In primary rat hepatocytes, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a decrease in DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor (CKI) proteins p21Cip-1/WAF1 and p16INK4a. To evaluate the relative importance of these CKIs in mediating this response, we determined the impact of prolonged MAPK activation on DNA synthesis in primary cultures of hepatocytes derived from mice embryonically deleted (null) for either p21Cip-1/WAF1 or p16INK4a. When MAPK was activated in wild-type mouse hepatocytes for 24 h, via infection with a construct to express an inducible oestrogen receptor–Raf-1 fusion protein (ΔRaf:ER), the expression of p21Cip-1/WAF1 and p16INK4a CKI proteins increased, cyclin-dependent kinase 2 (cdk2) and cdk4 activities decreased, and DNA synthesis decreased. Inhibition of RhoA GTPase function increased the basal expression of p21Cip-1/WAF1 and p27Kip-1 but not p16INK4a, and enhanced the ability of MAPK signalling to decrease DNA synthesis. Ablation of the expression of CCAATT enhancer-binding protein α (C/EBPα), but not of the expression of C/EBPβ, decreased the ability of MAPK signalling to induce p21Cip-1/WAF1. When MAPK was activated in p16INK4a-null hepatocytes for 24 h, the expression of p21Cip-1/WAF1 increased, cdk2 and cdk4 activities decreased and DNA synthesis decreased. In contrast with these findings, prolonged activation of the MAPK pathway in hepatocytes from p21Cip-1/WAF1-null mice enhanced cdk2 and cdk4 activities and caused a large increase in DNA synthesis, despite elevated expression of p16INK4a. Inhibition of RhoA GTPase activity in p21Cip-1/WAF1-null cells partly blunted both the basal levels of DNA synthesis and the ability of prolonged MAPK signalling to increase DNA synthesis. Expression of anti-sense p21Cip-1/WAF1 in either wild-type or p16INK4a-null hepatocytes decreased the ability of prolonged MAPK signalling to increase the expression of p21Cip-1/WAF1, and permitted MAPK signalling to increase both cdk2 and cdk4 activities and DNA synthesis. These results argue that the ability of prolonged MAPK signalling to inhibit DNA synthesis in hepatocytes requires the expression of p21Cip-1/WAF1, and that the increased expression of p16INK4a has a smaller role in the ability of this stimulus to mediate growth arrest. Our results also suggest that RhoA function can modulate DNA synthesis in primary hepatocytes via the expression of p21Cip-1/WAF1 and p27Kip-1.

scholarly journals Selective deficiency in protein kinase C isoenzyme expression and inadequacy in mitogen-activated protein kinase activation in cord blood T cells

2003 ◽  
Vol 370 (2) ◽  
pp. 497-503 ◽  
Author(s):  
Charles S.T. HII ◽  
Maurizio COSTABILE ◽  
George C. MAYNE ◽  
Channing J. DER ◽  
Andrew W. MURRAY ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Cord Blood ◽  
Map Kinase ◽  
Map Kinases ◽  
Mitogen Activated Protein Kinase ◽  
Biochemical Basis ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

The biochemical basis for the reduced lymphokine production by neonatal T cells compared with adult T cells remains poorly defined. Previous studies have raised the possibility that neonatal T cells could be deficient in their ability to transmit signals via protein kinase (PK) C. We now report that while PKC-dependent activation of the mitogen-activated protein (MAP) kinases, c-Jun N-terminal protein kinase and the extracellular signal-regulated protein kinase (ERK)1/ERK2, was deficient in cord blood T cells compared with adult blood T cells, marked activation of the MAP kinases in cord blood T cells was achieved via PKC-independent means. Consistent with a deficiency in the signalling capability of PKC, cord blood T cells were selectively deficient in the expression of PKCβI, ∊, θ and ζ. Stimulation of cord blood T cells resulted in a time-dependent increase in PKC expression, with increases detectable by 4h. This was accompanied by an enhancement in MAP kinase activation via PKC-dependent means. These novel data suggest that an inadequacy in PKC-MAP kinase signalling may be responsible, at least in part, for the phenotype of cord blood T cells.

scholarly journals Insulin regulation of mitogen-activated protein kinase kinase (MEK), mitogen-activated protein kinase and casein kinase in the cell nucleus: a possible role in the regulation of gene expression

1997 ◽  
Vol 323 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Sung-Jin KIM ◽  
Ronald C. KAHN
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Map Kinases ◽  
Nuclear Protein ◽  
Insulin Signalling ◽  
Receptor Activation ◽  
Casein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Intact Cells ◽  
Mitogen Activated Protein

After insulin receptor activation, many cytoplasmic enzymes, including mitogen-activated protein (MAP) kinase, MAP kinase kinase (MEK) and casein kinase II (CKII) are activated, but exactly how insulin signalling progresses to the nucleus remains poorly understood. In Chinese hamster ovary cells overexpressing human insulin receptors [CHO(Hirc)], MEK, CKII and the MAP kinases ERK I and ERK II can be detected by immunoblotting in the nucleus, as well as in the cytoplasm, in the unstimulated state. Nuclear localization of MAP kinase is also observed in 3T3-F442A adipocytes, NIH-3T3 cells and Fao hepatoma cells, whereas MEK is found in the nucleus only in Fao and CHO cells. Insulin treatment for 5–30 min induces a translocation of MEK from the cytoplasm to the nucleus, whereas the MAP kinases and CKII are not translocated into the nucleus in response to insulin during this period. However, nuclear MAP kinase and CKII activities increase by 2–3-fold within 1–10 min after stimulation with insulin. By using gel-shift assays, it has been shown that insulin also stimulates nuclear protein binding to an AP-1 site with kinetics similar to MEK translocation and MAP kinase and CKII activation. Treatment of the extracts in vitro with protein phosphatase 2A or treatment of the intact cells with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, a cell-permeable inhibitor of CKII, almost completely blocks the insulin-induced DNA-binding activity, whereas incubation of cells with a MEK inhibitor produces only a slight decrease. These results suggest that insulin signalling results in the activation of serine kinases in the nucleus via two pathways: (1) insulin stimulates the nuclear translocation of some kinases, such as MEK, which might directly phosphorylate nuclear protein substrates or activate other nuclear kinases, and (2) insulin activates nuclear kinases without translocation. The latter is true of CKII, which seems to regulate the binding of nuclear proteins to the AP-1 site, possibly by phosphorylation of AP-1 transcription factors.

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